The whole cell, supernatant and ribosome bound tRNAs have been isolated from SV40-transformed mouse fibroblasts and the acceptance activity for each of the 20 amino acids has been determined. The acceptance ratios of ribosome bound to whole cell tRNA are similar to unity (0 plus or minus 0.5) for most amino acids. However, the acceptance ratios for methionine, leucine and isoleucine are significantly above unity, and the acceptance ratios for glycine, histidine, proline, serine and tryptophan are below 0.5. The acceptance ratio of ribosome bound to whole cell tRNA for methionine is about 3.9. When the isoaccepting tRNA super Met are compared by reversed-phase cochromatography of C14-methionine ribosome bound tRNA and H4-methionine whole cell tRNA the acceptance ratios are 5.9 for MetI, 2.5 for MetII, and 1.4 for Met III. The pronounced enrichment for MetI, the most common isoaccepting tRNA in these cells, is in accordance with this species being the encaryotic cytoplasmic initiator tRNA. Further interpretation of the acceptance ratios for the remaining amino acids will also require comparison of the individual isoaccepting species after chromatographic separation. We conclude, however, that the method may prove useful in identifying other "special" tRNAs.